Lactic acid bacteria (LAB) are a group of taxonomically diverse, Gram-positive bacteria that are able to convert fermentable carbohydrates mainly into lactic acid, acidifying the growth medium in the process. In general, LAB species are best known for their use in the food industry, mainly in the preparation of fermented foods such as dairy products and certain kinds of meat. The commercial significance of the dairy fermentation industry, which encompasses production of e.g. cheese, yoghurt and sour cream, is well recognized worldwide.
Over the past decades, interest in LAB has dramatically increased. The fact that selected LAB strains can influence the intestinal physiology is widely recognised. L. lactis has enjoyed a growing interest as production host for heterologous proteins, and eventually as in situ production and delivery system for biologically active molecules (see below).
At present, much effort has been directed towards the use of genetically engineered (GM) LAB species as production and delivery tools for topical, mucosal administration of biological drugs, including cytokines, antibody fragments, growth factors, hormones and neuropeptides. (e.g. [6-12]). In particular the engineered food-grade bacterium Lactococcus lactis (L. lactis) was chosen as the preferred microorganism for the therapeutic delivery of biologically active polypeptides. Clearly, the concept of oral therapeutic protein delivery by engineered L. lactis strains opens exciting possibilities. A necessary attribute of any pharmaceutical product however is long-term stability (shelf life), typically at least 24 months under predefined storage conditions. To this end, an efficient, scalable and reliable manufacturing platform needs to be developed for engineered L. lactis-based Drug Substance (DS) and Drug Product (DP) formulations.
During manufacturing and subsequent storage, the critical parameter for product stability is long-term viability of the engineered bacteria (normally expressed as colony forming units (CFU) per gram in function of storage time). Manufacture, storage and eventual therapeutic use of LAB strains imposes significant stress on the bacteria [4]. On industrial settings, LAB may be preserved and distributed in liquid, spray-dried, frozen or lyophilized (freeze-dried) forms. While all these preparations can be suitable for use as starter cultures in the food industry, emphasis is increasingly being placed on long-term preservation methods that promote high cell viability and metabolic activity, as these parameters are considered a prerequisite for (bio)pharmaceutical applications. In order to maximize survival, addition of selected cryoprotectants to the biomass and subsequent lyophilization are crucial steps, especially considering the fact that viable and metabolically active bacteria are an absolute requirement to induce the desired therapeutic effect in situ.
Freeze-drying is widely regarded as one of the most suitable dehydration processes for bacteria, aiming to achieve a solid and stable final formulation [4]. It is one of the most common methods to store microbial cell cultures, even though survival rates after freeze-drying and during storage may vary between strains [5]. Survival after freeze-drying reflects the ability of the cells to resist the effects of rapid freezing and drying, such as membrane lipid oxidation and cell damage at several target sites [5]. It is well known that the freeze-drying of unprotected bacteria kills most of them, and those that survive, die rapidly upon storage. Several attempts have therefore been made to increase the number of surviving bacteria upon lyophilization and storage, with limited success (see below).
Lyophilization is by far the most frequently, if not exclusively used method to achieve long-term shelf life [16]. The choice of an appropriate drying medium/cryoprotectant mixture is critical to increase the survival rate of LAB during lyophilization and subsequent storage [4]. Several studies attempting to increase the survival rate of LAB during freeze-drying and/or subsequent storage have been reported (for review, see [4]). However, none of these publications demonstrate sufficient long-term stability (i.e. >80% survival after one year) of the freeze-dried bacteria, as required for pharmaceutical applications, in particular at room temperature (25° C.) or at 2-8° C.
For most LAB cultures of commercial interest for the dairy industry, skim milk powder is selected as drying medium because it stabilizes the cell membrane constituents, facilitates rehydration and forms a protective coating over the cells [4]. Supplementing skim milk with additional cryoprotectants agents may enhance its intrinsic protective effect.
Font de Valdez et al. describe the protective effect of adonitol in 10% skim milk, on 12 strains of LAB subjected to freeze-drying [17]. Although high survival rates during lyophilization are reported (ranging from 42-100%, depending on the strain), no data on long-term stability were provided. Castro et al. assessed the beneficial effects of skim milk (11%) or trehalose (5%) on the survival of Lactobacillus bulgaricus after freeze-drying, showing retention rates of 25% (viable cell count) compared to ˜1% in water alone [18]. Again, no data on stability during subsequent storage were reported.
Carvalho et al. (2003) demonstrated the stabilizing effect of either sorbitol or (mono)sodium glutamate (MSG), each added separately to LAB suspended in skim milk, on survival during lyophilization and subsequent storage for 3-6 months [19]. However, despite the fact that stability was increased compared to skim milk alone, the reported survival rates in the presence of sorbitol or MSG were still very low (<0.1%). Furthermore, long-term survival of the freeze-dried cells, stored in closed containers at 20° C. in air and kept in darkness for up to 8 months, showed a significant decrease of one or more logs over time.
Carcoba and Rodriguez studied the effects of various compounds, added individually to reconstituted skim milk (RSM), on cell survival and metabolic activity of L. lactis after freeze-drying [16]. They found that the sugars trehalose and sucrose, the polyols sorbitol and adonitol, as well as the amino acids β-alanine and glutamic acid, were capable of enhancing cell viability above the 44.3% recorded in RSM alone. However, actual survival rates with the supplemented media were not included, and no long-term storage data were disclosed.
As a final example, a study by Huang et al. developed and optimized a protective medium for Lactobacillus delbrueckii, resulting in a 86% cell viability after freeze-drying [20]. The composition of this medium was: sucrose 66.40 g/L, glycerol 101.20 g/L, sorbitol 113.00 g/L, and skim milk 130.00 g/L. Again, no long-term stability results were reported.
Huyghebaert et al. aimed to develop a freeze-dried powder formulation containing viable GM L. lactis bacteria with an acceptable shelf life [21]. To investigate the influence of the freeze-drying matrix, two different media were used; either M17 broth supplemented with 0.5% glucose (in order to obtain GM17), or 10% (w/v) skim milk supplemented with 0.5% glucose and 0.5% casein hydrolysate (in order to obtain GC-milk). Following freeze-drying, the influence of lyophilization parameters, freeze-drying matrix and different storage conditions was evaluated on short- and long-term viability.
When freeze-dried in conventional GM17 broth, absolute viability was less than 10%, while freeze-drying in GC-milk matrix resulted in significantly higher viability (60.0±18.0%). However, despite several attempts to standardize the freeze-drying procedure, significant batch-to-batch variability could not be avoided.
Short-term stability studies showed that viability already decreased ±20% after freeze-drying and storage for 1 week (GC-milk matrix). In long-term stability studies, relative viability was highly decreased after 1 month storage, followed by a logarithmic decrease during subsequent months of storage (GC-milk matrix, various storage conditions), indicating that long-term stability could not be achieved.
Considering the prior art in its entirety, it is obvious that skim milk is a recurrent component of freeze-drying media for LAB, and thus appears to be essential for bacterial viability. However, the use of milk derivatives in novel pharmaceutical compositions is strongly discouraged, especially in view of the Transmissible Spongiform Encephalopathy (TSE) risk associated with their use.
Next to high viability after production, freeze-dried LAB should also have an acceptable long-term shelf life for pharmaceutical applications. Stabilized dry bacterial compositions are for example described in US 2005/0100559, U.S. Pat. No. 3,897,307 and WO2004/065584. In US 2005/0100559 the dried bacterial composition are characterized in that they comprise a large fraction of stabilizers. See for example [055] in US 2005/0100559, wherein the stabilizers account for at least 40% (w/v). In WO2004/065584 sucrose or sucrose and maltodextrine were shown to improve the stability of a bacterial cell culture, but only at −20° C. In this reference there is no indication on how to improve the long-term shelf life (at room temperature) for a composition comprising freeze dried bacteria. In U.S. Pat. No. 3,897,307, all experiments start from a culture of different Lactobacillus species in nonfat milk that is subsequently freeze dried, optionally in the presence of stabilization potentiators selected from L-ascorbic acid, including edible salts thereof, and glutamic acid or aspartic acid, including the salts thereof. As such milk components are an important constituent of the stabilized dry bacterial compositions.
In other words none of the prior art addresses the replacement of the milk components with, sufficient survival and stability under long-term shelf storage. In fact, most of these studies lack precise data on initial viability, stability and bacterial density. Finally, none of them report on freeze-drying of GM bacteria and/or maintenance of their properties.